The Selection Of Reference Genes For Real-time RT-PCR In Apple(malus Domestica Borhk.) And The Development Of TaqMan Real-time RT-PCR Methods For Detecting Three Latent Viruses

Apple virus diseases can cause serious effect on the growth of apple trees and the quantity, quality of fruits. It’s hardly to eliminate the virus from the tree once infected and there is no effective chemical for controlling it. Cultivation and propagation of the virus-free seedling are one of the most effective measures for the control of virus diseases. The efficiently virus detection methods with strong specificity, high sensitivity and reliable reproducibility are the critical steps in the cultivation and propagation of virus-free seedling.In this study, four real-time RT-PCR methods with TaqMan probes were developed for the specific detection of Apple stem grooving virus(ASGV), Apple stem pitting virus(ASPV), Apple chlorotic leaf spot virus(ACLSV) and Actin gene while finding the Actin was the most stable reference gene in the different tissues of apple.The aim of this study was to provide efficient methods for researching the infection, multiplication, distribution of the three latent viruses. They also can be used to establish a multiplex real-time RT-PCR assay for detection and absolute quantitation the three latent viruses simultaneously. The main results were as follows:1. The expression of six reference genes(Actin, TUB, GAPDH, 18 SrRNA, UBQ, nad5) in five different tissues(bark, root, seed, immature and mature leaf) of apple were detected by real-time RT-PCR using SYBR green Ⅰ. The analysis with geNorm algorithms revealed that the stability of the six candidate reference genes in five different tissues from high to low was Actin = UBQ ﹥ GAPDH ﹥ nad5 ﹥ TUB ﹥18SrRNA. Thus, Actin and UBQ were the most suitable reference genes for apple.2. It was the first time to establish the real-time method RT-PCR with TaqMan probe for detecting Actin gene of apple. A pair of primers and a TaqMan probe were designed based on the conserved nucleotide sequence of Actin gene. The RNA standard of Actin was prepared with transcription in vitro to generate standard curve and the specificity, sensitivity and reproducibility of this method were evaluated. The results showed that the correlation coefficient(R2) of standard curve was 0.999 and the amplification efficiency(E) was 106.5%. The sensitivity of this method was 1.48×10 copies·μL-1 which was 1000 times higher than the conventional RT-PCR. The coefficients of variation between the intra- and inter-assay were both within 2.65%. This method could be used to detect Actin rapidly with strong specificity, high sensitivity and reliable reproducibility.3. A real-time RT-PCR method using TaqMan probe for detecting Apple stem grooving virus(ASGV) was developed in this study. A pair of primers and a TaqMan probe were designed based on the conserved nucleotide sequence of ASGV coat protein gene(cp). The recombinant plasmid of ASGV-cp was constructed as positive standard to generate standard curve and the specificity, sensitivity and reproducibility of this method were evaluated. The results showed that the correlation coefficient(R2) of standard curve was 0.999 and the amplification efficiency(E) was 96.8%. There was no crossing reaction with Apple stem pitting virus(ASPV), Apple chlorotic leaf spot virus(ACLSV) and Apple scar skin viroid(ASSVd), indicating a strong specificity. The sensitivity of this method was 10 copies·μL-1 which was 1000 times higher than the conventional RT-PCR. The coefficients of variation between the intra- and inter-assay were both within 1%. This method could be used to detect ASGV rapidly in practical samples with strong specificity, high sensitivity and reliable reproducibility.4. One real-time RT-PCR assay using TaqMan probe was developed for detecting ACLSV. A pair of primers and a TaqMan probe were designed based on the conserved nucleotide sequence of ACLSV coat protein(cp) gene, and the recombinant plasmid of ACLSV-cp was constructed as positive standard to generate standard curve. The specificity, sensitivity and reproducibility of this method were evaluated. The results showed that the correlation coefficient of standard curve was 0.999 and the amplification efficiency was 103.7%. There was no crossing reaction with ASGV, ASPV and ASSVd, indicating a strong specificity. The sensitivity of this method was 100 copies·μL-1 which was 100 times higher than the conventional RT-PCR. The coefficients of variation between the intra- and inter-assay were both within 0.84%. This method could be used to detect ACLSV rapidly in practical samples with strong specificity, high sensitivity and reliable reproducibility.5. This study established a TaqMan real-time RT-PCR method for detecting ASPV. A pair of primers and a TaqMan probe were designed based on the conserved nucleotide sequence of ASPV coat protein(cp) gene. The RNA standard of ASPV was prepared with transcription in vitro and the standard curve was plotted. The specificity, sensitivity and reproducibility of this method were evaluated. The results showed that the coefficient of determination(R2) was 0.996 and the amplification efficiency(E) was 99%. There was no crossing reaction with ASGV, ACLSV and ASSVd, indicating a strong specificity. The detection limit of this method was 1.31 × 102 copies·μL-1 and the sensitivity was 100 times higher than the conventional RT-PCR. The coefficients of variation between the intra- and inter-assay were both within 1.85%. This method could be used to detect ASPV rapidly and quantitatively in field samples with strong specificity, high sensitivity and reliable reproducibility.